The long term objective of the proposed project is to characterize new enterotoxic determinants potentially involved in the pathogenesis of Shigella flexneri diarrhea. The attenuated S. flexneri vaccine candidates tested so far have shown various degrees of reactogenicity, particularly watery diarrhea. Studies of the PI have resulted in the discovery of enterotoxins elaborated by S. flexneri that have not previously been described. These toxins may be responsible for the residual diarrhea observed during Shigella vaccine trials. The PI proposes to investigate these findings further in a blend of basic and applied studies which will yield fundamental insights into the pathogenesis of S. flexneri diarrhea. These findings will also provide crucial information that can be applied to engineer improved attenuated vaccines. Specific aims are: AIM 1: To purify a new enterotoxic factor of S. flexneri 2a which induces secretion both in vivo and in vitro in animal models. The PI has already partially purified this toxin, termed ShET1 for Shigella enterotoxin 1, and now intends to purify this moiety further and to raise specific monoclonal antibodies to it. AIM 2. To study the genetic regulation of ShET1 and to construct mutants of the set1 genes. The PI has identified, cloned, and sequenced the chromosomal region encoding this factor. He will now characterize these genes and their mechanisms of regulation. AIM 3.To establish the role of ShET1 in the pathogenesis of shigellosis and to study its mechanism of action. The PI will perform intestinal perfusion studies in human volunteers and, using an animal model, will determine ShET1 interaction with enterocyte receptors, its effects on transepithelial water and electrolyte transport, and the intracellular mediator(s) of its enterotoxic effect. AIM 4. To determine whether ShET1 and enteroinvasive Escherichia coli enterotoxin (EIET/ShET2), a second enterotoxin recently described by this group, share any functional similarities.